CN102296062A - Method for extracting genomic DNA of frozen semen of bull - Google Patents
Method for extracting genomic DNA of frozen semen of bull Download PDFInfo
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Abstract
The invention provides a method for extracting the genomic DNA of the frozen semen of bull, which comprises the steps of washing of the semen of the bull, lysis and digestion of the semen and purification of the genomic DNA. In the method, the digestion yield of the DNA of the semen of the bull is increased by improving the components and mixing ratio of digestive lysis solution and determining proper digestion time; meanwhile, instead of the conventional method for purification by organic solvent extraction, a simpler high-salt method is used to purify DNA, and the high-salt method has the advantages of using easily prepared reagent, effectively removing impurities, obtaining high-yield and high-quality genomic DNA of frozen semen, along with low cost and suitability for high-efficiency and batch preparation of genomic DNA of frozen semen.
Description
Technical field
The present invention relates to the extracting method of genomic dna, specifically, relate to the extracting method that a breeding oxen is frozen smart genomic dna.
Background technology
Holstein cow is the important domestic animal kind that dairy products are provided for the mankind, along with renewal and the raising of people to the milk preparation specification of quality, for meeting the need of market, need and improve the milk cow colony performance of giving milk to the milk cow seed selection.The realization of genetic breeding and technology of artificial insemination makes the seminal fluid of good bull be used to make and freezes essence (straw frozen semen or particle freeze essence) in a large number, is convenient to the propagation of its good heritability, improves colony's performance.Along with molecular biology and genetic development, carry out the seed selection of holstein cow and improve performance from the molecular biology angle, be the focus of current research.With respect to blood sample and ear tissue sample etc., it is many to freeze smart quantity, obtains easily and preserves, and is kind with the modal experiment material of bull.DNA is the carrier of genetic information, is most important biological information molecule, is the main object of molecular biology research.For it is checked order, the research of hybridization and expression of gene, it is very important obtaining high molecular and highly purified genomic dna.Operate two aspects that mainly comprise of extracting genome DNA: the one, and lysis and DNA discharge, and the 2nd, the purifying of DNA.This two aspect is determining genome to extract productive rate, integrity and the purification degrees of product.Exist animal different tissues genome in the market and extract test kit, modal is the related products of poba gene group DNA extraction, adopts the lysate composition and the purifying mode of innovation, has brought facility for the genomic extraction of blood sample.And with respect to freezing essence, rarely seen commercial test kit.It is to utilize the proteopepsis liquid digestion sperm that adds beta-mercaptoethanol that its genome extracts modal method, utilize method of organic solvent extraction purified product (the Sa nurse Brooker J of phenol/chloroform behind the released dna, Russell D.W. molecular cloning guide [M] Beijing, Science Press, 2002).There is following problem in actual applications in this method: extraction time is long, complex operation, and residual organic solvent influences subsequent experimental in the product, and it is big to freeze smart digestion difficulty, extracts the smart genomic dna of freezing of high-quality a large amount in batches for the laboratory and brings difficulty.
Mammalian sperm DNA is the carrier of genetic information, and is significant to producing offspring, and this special mission requires the chromatin of sperm to have special construction.Different with other cells, the chromatin of sperm forms to have passed through and doubles to concentrate and assembling (Jiang Huanhuan summary, Cao Yunxia, He Xiaojin examines and revises. staining of sperm matter integrity detection and auxiliary procreation technology. and international healthy reproduction/birth control magazine, 2011,1,30 (1): 58-62; Zou Yuping, Wang Xiaoquan, Lei Yiding, Pei Yanlong, Zhang Zhi's constitution. the extraction of several threatened plant and the nearly total DNA of edge monoid thereof and evaluation 3, Botany Gazette, 1994,36 (7): 528-533).The generation of mammalian sperm can be divided into spermatogonium, spermatocyte, spermatid three phases.In the spermatid stage, progressively replaced with DNA bonded histone (H1, H2a, H2b, H3, H4) in the nuclear by protamine, this process is called histone-protamine substitution reaction (histone-to-protamine replacement reaction, HPRR), (Wu Xiaofang, Chen Hui, Chen Song, Cao Jian, Fei Renren. the analysis of protamine mRNA in people and the big mouse sperm, reproduction and contraception, 2001,08,21 (4): 200-206).Protamine is that a kind of lower molecular weight basic protein (molecular weight is 4000-7000) contains 27-65 amino-acid residue, be rich in arginine (Arg) and halfcystine (Cys) (Zhao Xiangdong summary, Huang Yufeng examines and revises, the proteic progress of sperm nucleus, Journal of Andrology, 1998,11,4:38-41).Sperm DNA forms chromatin basic structure around protamine, and the disulfide linkage that the further oxidation of the outer sulfydryl of protamine intramolecularly then forms is extensively cross-linked, thereby karyomit(e) is concentrated gradually, and is more stable.Mature sperm examine this densification phenomenon to protection sperm genome sperm face the external world stress the time being kept perfectly property have vital role; (Jiang Huanhuan summarizes among the offspring can to guarantee complete being delivered to of genetic material; Cao Yunxia; He Xiaojin examines and revises; staining of sperm matter integrity detection and auxiliary procreation technology, international healthy reproduction/birth control magazine, 2011; 1,30 (1): 58-62).Exactly because this fine and close characteristic of mature sperm cell causes sperm fine and close more with respect to other histiocytic karyomit(e), genomic dna is not easy to break away from protamine.Make sperm DNA discharge difficulty in digestion, productive rate is low.In addition, freeze the dilution protective material that adds for spermoblast dilution, nutrition and protection are needed in the essence, compositions such as the carbohydrate that it contained, lipid and albumen bring obstacle also for the digestion and the genomic purifying of cell.
Summary of the invention
The purpose of this invention is to provide a breeding oxen and freeze the extracting method of smart genomic dna.
In order to realize the object of the invention, a breeding oxen of the present invention is frozen the extracting method of smart genomic dna, comprises the cleaning of spermatid, the cracking digestion of sperm and the purification step of DNA, wherein,
The cracking of sperm and digestion step are: add the spermatid lysate that freezes 2～3 times of volumes of smart stoste and 20w/v%SDS in the spermatid precipitation after cleaning and (make that the final concentration of SDS reaches 3%～5% in the system, w/v), leave standstill back vortex mixing, in mixed system, add Proteinase K (make Proteinase K concentration reaches 0.4mg/mL in the system) then, mixing, digested overnight; Wherein, the prescription of described spermatid lysate is: the EDTA0.01-0.0125mol/L of pH value 8.0, and the TrisCl 0.01-0.0125mol/L of pH value 8.0, SDS1%-1.25%, NaCl 2.9-3.625g/L, DTT 9.6-19.3g/L prepares with distilled water.Preferably, the prescription of described spermatid lysate is: the EDTA 0.0125mol/L of pH value 8.0, and the TrisCl 0.0125mol/L of pH value 8.0, SDS 1.25%, NaCl 3.625g/L, DTT9.625g/L prepares with distilled water.
The purification step of genomic dna is: the saturated aqueous common salt that adds 0.4～0.6 times of this digestion system cumulative volume in above-mentioned digestion system, leave standstill 5-10min behind the mixing, centrifugal, shift supernatant, and precipitate supernatant liquor with dehydrated alcohol, centrifugal after, washing precipitation, in 4 ℃ precipitation spent the night at last and be dissolved among TB storage liquid (available from TIANGEN Biotech (Beijing) Co., Ltd.) or the ddH2O-20 ℃ of preservations.
Preferably, the cracking of sperm and digestion step are in the preceding method: add spermatid lysate that freezes 2 times of volumes of smart stoste and the 20w/v%SDS that freezes 0.5 times of volume of smart stoste in the spermatid precipitation after cleaning, leave standstill back vortex mixing, in mixed system, add Proteinase K then, mixing, digested overnight.
Preferably, in the preceding method in the purification step of genomic dna be the saturated aqueous common salt that in the digestion system of spermatid, adds 0.6 times of this digestion system cumulative volume.
In the preceding method, the cleaning step of described bull sperm is: essence is frozen in control place centrifuge tube, add physiological saline, and the vortex mixing, centrifugal, must precipitate.
In the preceding method, described bull is frozen essence and freezes essence for the He Sitan bull.
The present invention also provides bull to freeze smart genome DNA extracting reagent kit, and it comprises:
The EDTA 0.01-0.0125mol/L of reagent I:pH value 8.0, the TrisCl0.01-0.0125mol/L of pH value 8.0, SDS 1%-1.25%, NaCl 2.9-3.625g/L, DTT9.6-19.3g/L prepares with distilled water; And reagent II: saturated aqueous common salt.
The aforementioned agents box also comprises: reagent III: physiological saline; Reagent IV:20%SDS; And reagent V: Proteinase K.
Preferably, described test kit comprises:
The EDTA 0.0125mol/L of reagent I:pH value 8.0, the TrisCl0.0125mol/L of pH value 8.0, SDS 1.25%, NaCl 3.625g/L, DTT 9.625g/L prepares with distilled water;
Reagent II: saturated aqueous common salt;
Reagent III: physiological saline;
Reagent IV:20w/v%SDS; And
Reagent V: Proteinase K.
The present invention further provides described test kit and freeze application in the smart genomic dna extracting the He Sitan bull.
Freeze some key issues that exist in the smart extracting genome DNA based on bull, the inventor improves digestion lysate composition and proportioning targetedly, determines suitable digestion time, improves the digestion productive rate of bull sperm DNA; Abandon the extractive purification process of original organic solvent simultaneously, adopt fairly simple high salt method to carry out the DNA purifying, the reagent of use is easy to preparation, and cost is low, can effectively remove impurity, obtains that productive rate height, matter are measured to freeze smart genome.Test kit of the present invention toxic substance easy to use, quick, that relate to is few, and the DNA amount that extracts is many, purity is high, is fit to the needs that Routine Test Lab is used.Can be widely used in and freeze efficiently with in batches obtaining of smart genomic dna.
Description of drawings
Fig. 1 freezes smart extracting genome DNA electrophoresis detection result for the He Sitan bull, and wherein M is DNA marker (7000,5500,3500,2000,1000,500bp), and 1-44 represents 44 samples.
Fig. 2 freezes smart genomic dna GHR gene purpose fragment PCR amplification for the He Sitan bull, and wherein M is DNA marker (600,500,400,300,200,100bp), and 1-44 represents 44 samples.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment, the raw materials used commercial goods that is.Unless otherwise indicated, the preparation of the solution among the embodiment is all carried out under 20 ℃.
The extraction that embodiment 1 He Sitan bull is frozen smart genomic dna
1 material and reagent
Physiological saline (0.9%): take by weighing 0.9 gram sodium-chlor, be dissolved in a small amount of distilled water, be diluted to 100 milliliters.Autoclaving is standby.
Spermatid cracking and digestion reagent:
(1) spermatid lysate: sterile purified water 60mL, add 0.5mol/LEDTA (pH=8.0) 2mL, 1mol/L TrisCl (pH=8.0) 1mL and 10%SDS 10mL, 0.29g NaCl, 0.77g DTT (can double to add DTT for the indigestibility sample) adds the water constant volume to 80mL behind the mixing.Liquid mixture prepared normal temperature is preserved, and can not surpass a week duration of service.
0.5mol/L add 18.61g two water disodium ethylene diamine tetraacetate (EDTA-Na2H in the EDTA:80mL distilled water
2O), with NaOH regulator solution pH value to 8.0 (about 20gNaOH particle), add water then and be settled to 100mL behind the mixing, autoclaving is standby after the packing.
Add 121.1g Tris alkali in the 1mol/L TrisCl:800mL distilled water, transfer pH value of solution value to 8.0 (solution can be set up the pH value after being chilled to room temperature at last), add water then and be settled to 1L, autoclaving after the packing with hydrochloric acid.When solution becomes yellow, should abandon.
Add 100g SDS in the 10%SDS:900mL distilled water, be heated to 68 ℃ and use the magnetic stirrer hydrotropy, with HCl regulator solution pH value to 7.2, add water and be settled to 1L behind the mixing.Room temperature preservation need not sterilization.
(2) spermatid Digestive system: 400 μ L spermatid lysates, 100 μ L 20%SDS, 10 μ L Proteinase Ks.
Add 200g SDS in 20% the SDS:900mL distilled water, be heated to 68 ℃ and use the magnetic stirrer hydrotropy, with HCl regulator solution pH value to 7.2, add water and be settled to 1L behind the mixing.Room temperature preservation need not sterilization.As find solution solidifies, illustrate that room temperature is low, uses after the heating for dissolving again.
Proteinase K (20 mg/mL): TIANGEN Biotech (Beijing) Co., Ltd., article No. RT403-01.
The DNA purified reagent:
Saturated aqueous common salt: in the time of 20 ℃, saturated sodium-chloride concentration is 26.47%, and promptly dissolving 36 restrains sodium-chlor in 100mL water, and appropriate supplement adds sodium-chlor, and mixing still has solid not molten after leaving standstill 10-20min, and the gained supernatant is saturated aqueous common salt.
75% ethanol: get 3 volume dehydrated alcohols and 1 volume distilled water, mixing is standby.
Agarose horizontal strip electrophoresis detection reagent:
50 * TAE:Tris alkali 242g, glacial acetic acid 57.1mL, 0.5mol/L EDTA (pH=8.0) 100mL add water and fully dissolve, and are settled to 1L.
Bromination second pyridine (EB) stock solution when sample (on the electrophoresis use): the pyridine of 1g bromination second is dissolved in the 100mL distilled water.
1% sepharose that electrophoresis uses is prepared with 50 * TAE liquid.Agarose dry powder wins Tai Boke skill company available from east, Beijing.
The extraction that smart genomic dna is frozen in 2 He Sitan bull control
2.1 essence is frozen in bull control
Adopt the control of 44 He Sitan breeding oxens to freeze essence (available from Beijing Milk Cow Center).
Smart volume is frozen in control: 250 μ l/ pipe, the every dosage of regulation freezes essence thaw back motility of sperm 〉=35%, the sperm count that advances 〉=8,000,000 among the GB4143-2008.Every dosage freezes smart total sperm count and is about about 2,000 ten thousand (total sperm count with season, an ox situation, to freeze factor mobility scale such as smart manufacturer bigger).
Employed dilution protective material in the essence is frozen in preparation control, adopts the prescription A or the B that recommend among the GB4143-84, and its compound method is as follows:
Prescription A: Trisodium Citrate, fructose, yolk diluent:
A liquid: 100 milliliters of distilled water, Trisodium Citrate 2.97 grams, 10 milliliters in yolk;
B liquid: get 41.75 milliliters of a liquid, add fructose 2.5 grams, 7 milliliters of glycerine, mixing is promptly.
Prescription B: skimmed milk, yolk diluent:
82 milliliters of skimmed milks, 10 milliliters of b liquid, 8 milliliters of glycerine, mixing is promptly.
2.2 freeze the extraction of smart genomic dna
Step 1: get the 2mL centrifuge tube, put into about 200 μ L volumes and freeze smart stoste, add 500 μ L physiological saline, vortex mixing, the centrifugal 1min of 12000rpm.Remove supernatant liquor (refining and the diluent in the seminal fluid frozen in removal control), keep the spermatid precipitation.
Step 2: repeating step 1 once.
Step 3: add 400 μ L spermatid lysates and 100 μ L 20%SDS to precipitation, leave standstill vortex mixing precipitation and solution behind the 2-3min.In mixed solution, add 10 μ L Proteinase Ks then, put upside down mixing.
Step 4: the sample of mixing is put into 56 ℃ water-bath, digested overnight (about 10 hours, the indigestibility sample suitably increases digestion time).
Step 5: the sample that cancellationization is good, add 300 μ L saturated aqueous common salts, put upside down 2-3min, put into 4 ℃ of refrigerators and leave standstill 5-10min, albumen and SDS mixture in the precipitation solution.The centrifugal 10min of 12000rpm moves on to supernatant (DNA is present in the supernatant) in the new pipe then.
Step 6: add the iced dehydrated alcohol of 1000 μ L to supernatant liquor.Put upside down 1-2min, flocks (DNA precipitation) occurs, then the centrifugal 2min of 12000rpm.The visible DNA precipitation of naked eyes is affixed on the centrifuge tube bottom.Remove supernatant liquor, keep precipitation.
Step 7: in centrifuge tube, add the ethanol of 500 μ L 75%, put upside down mixing, clean residual impurity and salt ion on the DNA precipitation.The centrifugal 2min of 12000 rpm removes supernatant then, keeps precipitation.
Step 8: repeating step 7 once.
Step 9: uncap and place 2-3min, make residual ethanol volatilization on the DNA precipitation.
Step 10: (TIANGEN Biotech (Beijing) Co., Ltd. also can use ddH to add 200 μ L TB in centrifuge tube
2O guarantees solution submergence DNA precipitation, and room temperature is placed and spent night half an hour or 4, treats the resolution of precipitate genome quality of back Detection and Extraction fully.
2.3 extract the genome quality examination of purifying
Horizontal strip electrophoresis detects: the agarose horizontal strip electrophoresis with 1% detects, and puts 2 μ L samples, 130V electrophoresis 10min.Detect the integrity and the purity of DNA band.Electrophoresis detection result as shown in Figure 1, as can be seen from Figure 1, the band of 44 samples shows and to be tangible master tape, does not take off the tail phenomenon, illustrates that the DNA integrity is good; The point sample hole is clean, does not have albumen residual; Band is clear, and brightness is strong, illustrates that the DNA concentration of extracting is higher.
Spectrophotometer detects:
Adopt Thermo Scientific NanoDrop 2000 spectrophotometers, measure absorbance value at wavelength 230nm, 260nm and 280nm place, absorbance value according to 260nm calculates the DNA productive rate, according to the purity of 260nm/280nm and 260nm/280nm photoabsorption ratio judgement DNA sample.Detected result is as shown in table 1.
Table 1
Bull of the present invention is frozen smart genome DNA extracting reagent kit, and it comprises:
Scheme one:
The EDTA 0.01-0.0125mol/L of reagent I:pH value 8.0, the TrisCl0.01mol/L of pH value 8.0, SDS 1%, NaCl 2.9g/L, DTT 9.6g/L prepares with distilled water; And reagent II: saturated aqueous common salt.
Scheme two:
The EDTA 0.0125mol/L of reagent I:pH value 8.0, the TrisCl0.0125mol/L of pH value 8.0, SDS 1.25%, NaCl 3.625g/L, DTT 19.3g/L prepares with distilled water; And reagent II: saturated aqueous common salt.
Scheme three:
The EDTA 0.01mol/L of reagent I:pH value 8.0, the TrisCl0.0125mol/L of pH value 8.0, SDS 1.25%, NaCl 3.6g/L, DTT 19.3g/L prepares with distilled water; And reagent II: saturated aqueous common salt.
Scheme four:
The EDTA 0.0125mol/L of reagent I:pH value 8.0, the TrisCl0.01mol/L of pH value 8.0, SDS 1.25%, NaCl 2.9g/L, DTT 19.3g/L prepares with distilled water; And reagent II: saturated aqueous common salt.
Scheme five:
The EDTA 0.011mol/L of reagent I:pH value 8.0, the TrisCl0.012mol/L of pH value 8.0, SDS 1.2%, NaCl 3g/L, DTT 16g/L prepares with distilled water; And reagent II: saturated aqueous common salt.
Scheme six:
The EDTA 0.012mol/L of reagent I:pH value 8.0, the TrisCl0.01mol/L of pH value 8.0, SDS 1%, NaCl 3.5g/L, DTT 10g/L prepares with distilled water; And reagent II: saturated aqueous common salt.
Scheme seven:
The EDTA 0.0125mol/L of reagent I:pH value 8.0, the TrisCl0.011mol/L of pH value 8.0, SDS 1.1%, NaCl 2.9g/L, DTT 9.5g/L prepares with distilled water; And reagent II: saturated aqueous common salt.
Scheme eight:
The EDTA 0.0125mol/L of reagent I:pH value 8.0, the TrisCl0.0125mol/L of pH value 8.0, SDS 1.25%, NaCl 3.625g/L, DTT 9.625g/L prepares with distilled water;
Reagent II: saturated aqueous common salt;
Reagent III: physiological saline;
Reagent IV:20%SDS; And
Reagent V: Proteinase K.
The application that the bull that experimental example adopts the inventive method to extract is frozen smart genomic dna
The amplification of GHR gene purpose fragment PCR:
The segmental amplimer sequence of GHR gene purpose is F:5 '-AATACTTGGGCTAGCAGTGACAATAT-3 ' and R:5 '-ACTGGGTTGATGAAACACTTCACTC-3 ', and product length is 175bp, and reaction system and condition are as follows.
PCR reaction system (20 μ L): 10 * amplification buffer, 2 μ L, 4 kinds of dNTP mixtures (2.5mmol/L), 1.6 μ L, primers F (20pmol/ μ L) 0.4 μ l, primer R (20pmol/ μ L) 0.4 μ L, template DNA freezes smart genomic dna (100ng/ μ L) 1 μ L for the He Sitan bull that obtains among the embodiment 1, Taq archaeal dna polymerase (5U/ μ L) 0.2 μ L, ddH
2O 14 μ L (damping fluid, dNTP, polysaccharase are available from TaKaRa company).
The PCR reaction conditions: 94 ℃ of pre-sex change of 5min, 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ of extension 30s repeat 2-4 step 35 circulations, and 72 ℃ are continued to extend 7min then.2% agarose gel electrophoresis detects the PCR product.Amplified production electrophoresis detection result as shown in Figure 2, from this result as can be seen, the purpose band is clear bright, illustrate that template quality is good, is suitable for increasing.
By electrophoretic spectrophotometric detection, on band integrity, concentration and purity, 44 samples that single job is extracted can both satisfy the specification of quality that molecule is tested genomic dna, and the segmental amplification of GHR purpose has also proved this point.260/230 ratio of individual samples is high slightly, may be that salt ion is residual, can be with the sample DNA redeposition, and after the washing once of 75% alcohol, residue is removed in dissolving again.To extract sample and be used for the SNPs chip and carry out individual somatotype (the BovineSNP50 BeadChip of Illumina company chip), the genome DNA sample quality inspection is qualified, can be used for the somatotype experiment.Sample is deposited 1 month and-20 ℃ after frozen 1 year at 4 ℃, and carrying out horizontal strip electrophoresis, to detect the show sample band complete, and it is not obvious to degrade.
Though above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. a breeding oxen is frozen the extracting method of smart genomic dna, comprises the cleaning of spermatid, the cracking of sperm and the purification step of digestion and DNA, it is characterized in that,
The cracking of sperm and digestion step are: add spermatid lysate and the 20w/v%SDS that freezes 2～3 times of volumes of smart stoste in the spermatid precipitation after cleaning, leave standstill back vortex mixing, add Proteinase K, mixing, digested overnight then in mixed system; Wherein, the prescription of described spermatid lysate is: the EDTA 0.010-0.0125mol/L of pH value 8.0, the TrisCl 0.01-0.0125mol/L of pH value 8.0, SDS 1%-1.25%, NaCl 2.9-3.625g/L, DTT9.6-19.3g/L prepares with distilled water;
The purification step of genomic dna is: the saturated aqueous common salt that adds 0.4～0.6 times of this digestion system cumulative volume in above-mentioned digestion system, leave standstill 5-10min behind the mixing, centrifugal, shift supernatant, and precipitate supernatant liquor with dehydrated alcohol, centrifugal after, washing precipitation, at last precipitation is dissolved in the distilled water-20 ℃ of preservations.
2. method according to claim 1 is characterized in that, the prescription of described spermatid lysate is: the EDTA 0.0125mol/L of pH value 8.0, the TrisCl0.0125mol/L of pH value 8.0, SDS 1.25%, NaCl 3.625g/L, DTT 9.625g/L prepares with distilled water.
3. method according to claim 1 and 2, it is characterized in that, the cracking of sperm and digestion step are: add spermatid lysate that freezes 2 times of volumes of smart stoste and the 20w/v%SDS that freezes 0.5 times of volume of smart stoste in the spermatid precipitation after cleaning, leave standstill back vortex mixing, in mixed system, add Proteinase K then, make that Proteinase K concentration reaches 0.4mg/mL in the system, mixing, digested overnight.
4. method according to claim 1 and 2 is characterized in that, is the saturated aqueous common salt that adds 0.6 times of this digestion system cumulative volume in the digestion system of spermatid in the purification step of genomic dna.
5. method according to claim 1 and 2 is characterized in that, the cleaning step of bull sperm is: will freeze essence and place centrifuge tube, add physiological saline, and the vortex mixing, centrifugal, must precipitate.
6. method according to claim 1 and 2 is characterized in that, described bull is frozen essence and freezes essence for the He Sitan bull.
7. bull is frozen smart genome DNA extracting reagent kit, it is characterized in that, it comprises:
The EDTA 0.010-0.0125mol/L of reagent I:pH value 8.0, the TrisCl0.01-0.0125mol/L of pH value 8.0, SDS 1%-1.25%, NaCl 2.9-3.625g/L, DTT9.6-19.3g/L prepares with distilled water;
Reagent II: saturated aqueous common salt.
8. test kit according to claim 7 is characterized in that it also comprises:
Reagent III: physiological saline;
Reagent IV:20w/v%SDS;
Reagent V: Proteinase K.
9. test kit according to claim 8 is characterized in that it comprises:
The EDTA 0.0125mol/L of reagent I:pH value 8.0, the TrisCl0.0125mol/L of pH value 8.0, SDS 1.25%, NaCl 3.625g/L, DTT 9.625g/L prepares with distilled water;
Reagent II: saturated aqueous common salt;
Reagent III: physiological saline;
Reagent IV:20w/v%SDS;
Reagent V: Proteinase K.
10. each described test kit of claim 7-9 freezes application in the smart genomic dna extracting the He Sitan bull.
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| CN108384781A (en) * | 2018-05-22 | 2018-08-10 | 江苏集萃药康生物科技有限公司 | Mouse sperm genome DNA extracting method |
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